PRE-STEADY-STATE KINETIC-STUDY OF THE EFFECTS OF K-MEMBRANE CA2+-ATPASE( ON THE PARTIAL REACTIONS OF THE CATALYTIC CYCLE OF THE PLASMA)

The effects of 100 mM K+ on the partial reactions that take place duri ng ATP hydrolysis by the calcium ion-dependent ATPase from plasma memb rane (PM-Ca2+-ATPase) were studied at 37 degrees C on fragmented intac t membranes from pig red cells by means of a rapid chemical quenching technique. At 10 mu M [gamma-P-32]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140 %, to 8 4 +/- 11 s(-1) and the steady-state level of phosphoenzyme (EP) by 25 %, to 3.4 +/- 0.17 pmol/mg of protein. If added together with [gamma-P -32]ATP at the beginning of phosphorylation, K+ was much less effectiv e than if added earlier, indicating that it did not act on the phospho rylation reaction. Measurements of the E(2) --> E(1) transition by pho sphorylation showed that in medium with Ca2+ and Mg2+, K+ increased th e k(app) of the transition by 55 %, to 14 +/- 3 s(-1) and the apparent concentration of E(1) by 45 %, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubat ed with K+. The presence of K+ did not change the slow decay of EP wit hout Mg2+ but activated the decay of EP made with Mg2+, increasing its k(app) by 60 %, to 91 +/- 12 s(-1). In contrast with observations mad e during phosphorylation, if added at the beginning of dephosphorylati on K+ was fully effective in favouring decomposition of EP made in med ium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP p lus calmodulin, which activate hydrolysis of CaE(2)P, the effect of K on dephosphorylation was conserved. Because the sites for K+ are intr acellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca2+-ATPase under physiological conditi ons.